This research has two major aims. The first is to enhance the utility of T4 RNA ligase as a reagent for the synthesis and modification of nucleic acids by enlarging our understanding of its enzymological properties. a particular goal is to improve its ability to use DNA substrates so that it can be used to synthsize defined sequences of DNA. We propose to analyze reaction mixtures for each of the nucleotide, oligonucleotide, and enzyme species present under various conditions. The second major aim is to contribute to our understanding of specific protein-nucleic acid interactions by synthesizing a series of oligodeosyribonucleotides containing variations of the Eco RI restriction endonuclease and methylase recognition sequence, d(G-A-A-T-T-C). Individual syntherized sequences will contain single base analogue substitutions at define sites. Incorporation of Ura, 5-BrUra, 5-Mecyt, 5-BrCyt, 2-aminopurine, 2,6-diaminopurine, N(6)-MeAde, 3-deazaAde, and 7-deaza-8-azaGua will allow systematic alteration of may of the possible contact points between the enzymes and the major and minor grooves of the DNA. The two enzymes will be examined for their ability to interact with the modified substrates.